The vertebrate immune response to infection begins with the recognition by the innate immune system of conserved molecular signatures of pathogens, known as PAMPs (Pathogen Associated Molecular Patterns), provoking an immediate and often massive inflammatory response. The innate response holds the pathogen in check, but also plays a crucial role in the generation of acquired immunity. The recognition of PAMPs by the innate system is mediated by a number of receptors, of which the Toll-like Receptors (TLRs) play a prominent role. The molecular basis for the recognition of PAMPs by TLRs is a main interest of my laboratory. In collaboration with Dr. David Davies (LMB, NIDDK) and with the help of the Protein Expression Laboratory (SAIC/Frederick), we expressed mg amounts of the extracellular domain (ECD) of TLR3 and we continue to develop ways of expressing ECDs of other TLRs. The ECD of TLR3 crystallized and we determined its structure by X-ray crystallography, with and without its ligand, dsRNA. The structure of the TLR3-ECD consists of a 23 turn coil, bent into the shape of a horseshoe. The molecule is heavily glycosylated, except that one lateral face of the horseshoe is totally devoid of glycan. In solution, purified TLR3-ECD binds dsRNA at mildly acidic pH with an affinity that increases with dsRNA length. TLR3-ECD is monomeric in solution, but it forms dimers when bound to dsRNA. These dimers bind to 45 bp segments of dsRNA and multiple TLR3-ECD dimers bind to long dsRNAs. To determine the molecular basis for ligand binding and signaling, we isolated, crystallized, and solved the structure of the TLR3 signaling complex, consisting of two TLR3-ECD molecules bound to one 46 bp dsRNA oligonucleotide. The glycan-free surfaces of two TLR3-ECDs in the complex face one another on opposite sides of the dsRNA molecule which lies between them. The overall structure of mTLR3-ECD:dsRNA complex contains a two-fold symmetry axis, dictated by the inherent two fold symmetry of the ligand. No conformational change was observed in either the receptor or the ligand upon ligand binding, suggesting that dimerization per se is the activation signal for TLR3. Three intermolecular contacts on the glycan free surfaces of the two TLR3-ECDs stabilize the complex, two protein-dsRNA interactions, and one homotypic interaction between the two TLR3-ECD molecules, that is responsible for dimer formation. Using a novel ELISA to analyze dsRNA binding by mutant TLR3 constructs we identified the essential interacting residues and showed that the simultaneous interaction of all three sites is required for ligand binding. In addition, we found that TLR3 is unable to bind dsRNA when dimerization is prevented by mutating residues in the dimerization site or by immobilizing TLR3 at low density. Our results indicate that dimerization of TLR3 is essential for ligand binding and that the three TLR3 contact sites individually interact weakly with their binding partners but together form a high affinity ligand-receptor complex. The three other endosomal TLRs, TLRs 7, 8, and 9 form a closely related family of TLR paralogs that, like TLR3, recognize nucleic acid PAMPs, but differ from TLR3 in primary structure. It can be surmised from their amino acid sequences that these TLRs have several extended loops protruding from the glycan-free and convex surfaces of the LRR horseshoe, and an extended undefined, poorly conserved region between LRRs 14 and 15. Unfortunately the ECDs of these TLRs are secreted from insect cells in very low amounts, and the secreted material forms large, undefined aggregates. Currently we are investigating TLR9, which recognizes DNA that contains unmethylated CpG sequences. Our current hypothesis is that TLR9-ECD forms a complex with other proteins in vivo, and this complex recognizes CpG DNA. We have previously shown that most TLR9 resides in the ER, then migrates in small amounts to endosomes where it encounters its ligand. We found that TLR9 interacts with the gp96 chaperone and with Prat4A, a co-chaperone, as expected for ER proteins. Our immediate goal is it to obtain cells in which most of the TLR9 has migrated to endosomes, and then to examine the endosomal TLR9, both full length and ECD for possible proteins that form a complex with it. We have been partially successful in enhancing the amount of endosomal TLR9 through the use of inhibitors that block protein synthesis and TLR9 degradation, and we are employing several strategies to improve upon these results. We are also trying to piece together the TLR9 structure by making chimeras that contain parts of TLR9 grafted onto the TLR3 framework.